Beta actin antibody

PDAC cell lines (300,000 cells per well, 6-well plate) and frozen tissue sections of orthotopic tumor xenografts and normal adjacent tissue were pretreated with either 5 µM cathepsin inhibitor (GB111^{34 (link)}, not fluorescently labeled) or DMSO (0.1%). Then, all samples were incubated for 4 h with Cy5-labeled activity-based probe GB123^{33 (link)} (0.25 µM for tissues or 2 µM for cells, 0.1% DMSO final concentration). To visualize active cathepsin labeling, tissue sections were stained with DAPI and imaged with a fluorescent microscope equipped with a 630 nm laser. Treated PDAC cells were lysed, separated on 12.5% SDS-PAGE and scanned by Typhoon scanner (GE Healthcare) using an excitation/emission filter set of 635/670 nm. Following scanning, SDS–PAGE was immunoblotted on a PVDF membrane and incubated with beta-Actin antibody (Abcam, 1:1000). Chemiluminescence was measured with a ChemiDocXRS imaging system.

The cells were washed with ice-cold PBS, and the proteins were extracted using RIPA buffer supplemented with protease and phosphatase inhibitor cocktails (5892970001 and 4906837001, Roche, Switzerland). The lysates were centrifuged at 13,000 rpm for 5 min, and supernatants were quantified using Bradford reagent. Western blotting was performed using p44/42 MAPK antibody (4695S, CST, USA), phospho-p44/42 MAPK antibody (4370S, CST, USA) and beta actin antibody (ab8226, Abcam, UK), and western blot images were analysed with ImageQuant LAS 3000 (GE Healthcare, USA).

Three donors from each chondrogenic group were analysed by western blot. The protein input was 35 μg/lane in TruPage gels (Cat. no. PCG2004; Sigma-Aldrich). The protein was separated along with a BLUeye Prestained Protein Ladder (Cat. no. PM007–0500; Sigma-Aldrich) and MagicMark™ XP Western Protein Standard Ladder (Cat. no. LC5602; Novex). Proteins were transferred to a PVDF membrane, blocked for 2 h in PBS-Tween (0.05%) buffer containing BSA (2%) and incubated with 0.1 μg/mL of prolyl 4-hydroxylase 1 antibody (P4HA1; Cat. no. NB100–57852; Novus Biologicals) overnight at 4 °C. The membrane was incubated with secondary donkey anti-goat antibody (Cat. no. HAF109; Novus Biologicals) for 1 h at room temperature. Finally, a chemiluminescence detection solution (Cat. no. 170–5040, BioRad) was applied to the membrane before acquiring the images using an ImageQuant LAS 4000 CCD camera. Beta-actin antibody (Cat. no. AB8227; Abcam) and goat anti-rabbit antibody (Cat. no. AB6721; Abcam) were used as loading control and secondary antibody for beta-actin, respectively. Relative density was assessed using ImageJ software to compare the chondrogenic groups.